Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes. In this study, we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and fucosylated O-glycans for L-selectin binding. The functional importance of these modifications was determined by analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at Nterminal Thr or Tyr residues. Simultaneous expression of core-2 1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and Pselectin interactions with PSGL-1 further supported these observations. Additional experiments showed that core-2 O-glycans attached to Thr-57 were also of critical importance in regulating the velocity and stability of leukocyte rolling. These observations pinpoint the structural characteristics of PSGL-1 that are required for optimal interactions with L-selectin and may be responsible for the specific kinetic and mechanical bond properties of the L-selectin-PSGL-1 adhesion receptorcounterreceptor pair.Selectins play a major role in regulating leukocyte migration in inflammatory lesions by mediating leukocyte rolling along vascular wall at site of inflammation (1-8). L-selectin is expressed by most leukocytes whereas P-selectin and E-selectin expression is induced on activated platelets and/or endothelial cells (1,2,4,5,7,9). E-, P-, and L-selectin function at different although overlapping phases of the inflammatory reaction (10).P-selectin interacts with its major ligand P-selectin glycoprotein ligand-1 (PSGL-1) 1 and supports leukocyte rolling along postcapillary venules at the earliest phase of inflammation (11-13). Several studies have indicated that L-selectin mediates both primary leukocyte-endothelial interactions (14, 15) and secondary interactions between circulating and adherent leukocytes, which both participate in leukocyte recruitment in inflammatory lesions. Secondary interactions are mainly supported by the interaction of PSGL-1 with L-selectin (16 -18).PSGL-1 is a mucin-like glycoprotein expressed as a homodimer on leukocyte microvilli (4, 19 -21). P-selectin binds with relatively high affinity (K d ϳ300 nM) (22) to PSGL-1 by reacting with N-terminal tyrosine sulfate residues and with the sLe x tetrasaccharide determinants presented...